Extraction of niclosamide from commercial approved tablets into aqueous buffered solution creates potentially approvable oral and nasal sprays against COVID-19 and other respiratory infections

Motivation The low solubility, weak acid drug, niclosamide is a host cell modulator with broad-spectrum anti-viral cell-activity against many viruses, including stopping the SARS-CoV-2 virus from infecting cells in cell culture. As a result, a simple universal nasal spray preventative was proposed and investigated in earlier work regarding the dissolution of niclosamide into simple buffers. However, starting with pharmaceutical grade, niclosamide represents a new 505(b)(2) application. The motivation for this second paper in the series was therefore to explore if and to what extent niclosamide could be extracted from commercially available and regulatory-approved niclosamide oral tablets that could serve as a preventative nasal spray and an early treatment oral/throat spray, with possibly more expeditious testing and regulatory approval. Experimental Measurements of supernatant niclosamide concentrations were made by calibrated UV-Vis for the dissolution of niclosamide from commercially available Yomesan crushed into a powder for dissolution into Tris Buffer (TB) solutions. Parameters tested were as follows: time (0–2 days), concentration (300 µM to -1 mM), pH (7.41 to 9.35), and anhydrous/hydrated state. Optical microscopy was used to view the morphologies of the initial crushed powder, and the dissolving and equilibrating undissolved excess particles to detect morphologic changes that might occur. Results Concentration dependence: Niclosamide was readily extracted from powdered Yomesan at pH 9.34 TB at starting Yomesan niclosamide equivalents concentrations of 300 µM, 600 µM, and 1 mM. Peak dissolved niclosamide supernatant concentrations of 264 µM, 216 µM, and 172 µM were achieved in 1 h, 1 h, and 3 h respectively. These peaks though were followed by a reduction in supernatant concentration to an average of 112.3 µM ± 28.4 µM after overnight stir on day 2. pH dependence: For nominal pHs of 7.41, 8.35, 8.85, and 9.35, peak niclosamide concentrations were 4 µM, 22.4 µM, 96.2 µM, and 215.8 µM, respectively. Similarly, the day 2 values all reduced to 3 µM, 12.9 µM, 35.1 µM, and 112.3 µM. A heat-treatment to 200 °C dehydrated the niclosamide and showed a high 3 h concentration (262 µM) and the least day-2 reduction (to 229 µM). This indicated that the presence, or formation during exposure to buffer, of lower solubility polymorphs was responsible for the reductions in total solubilities. These morphologic changes were confirmed by optical microscopy that showed initially featureless particulate-aggregates of niclosamide could grow multiple needle-shaped crystals and form needle masses, especially in the presence of Tris-buffered sodium chloride, where new red needles were rapidly made. Scale up: A scaled-up 1 L solution of niclosamide was made achieving 165 µM supernatant niclosamide in 3 h by dissolution of just one fifth (100 mg niclosamide) of a Yomesan tablet. Conclusion These comprehensive results provide a guide as to how to utilize commercially available and approved tablets of niclosamide to generate aqueous niclosamide solutions from a simple dissolution protocol. As shown here, just one 4-tablet pack of Yomesan could readily make 165 L of a 20 µM niclosamide solution giving 16,500 10 mL bottles. One million bottles, from just 60 packs of Yomesan, would provide 100 million single spray doses for distribution to mitigate a host of respiratory infections as a universal preventative-nasal and early treatment oral/throat sprays throughout the world. Graphical Abstract pH dependence of niclosamide extraction from crushed Yomesan tablet material into Tris buffer (yellow-green in vial) and Tris-buffered saline solution (orange-red in vial). Initial anhydrous dissolution concentration is reduced by overnight stirring to likely monohydrate niclosamide; and is even lower if in TBSS forming new niclosamide sodium needle crystals grown from the original particles. Supplementary Information The online version contains supplementary material available at 10.1186/s41120-023-00072-x.


Supplemental Information
Supplemental information provides added and underlying data for the main text. It includes: • UV-Vis absorbance measurements that conformed the instrument resolution (Photometric accuracy) • UV-Vis absorbance spectra of pure vanillin as a function of its concentration and pH of Tris buffer that identify it as the absorbing "impurity" in the Yomesan powder • UV-Vis absorbance spectra Talc and Cornstarch that show they did not affect the results • The measured peak maximum niclosamide concentrations and % niclosamide that were extracted by dissolution of Yomesan tablet powder in nominal pH 9.35 Tris buffer • The initial rates of dissolution data in Figs. 4  included because, if used as an oral spray, it would essentially be applied locally to the same buccal and throat epithelium as the "thoroughly chewed" 2 grams of Yomesan tablets. The amount per 100uL sprayed dose though would be 5.4 micrograms for a 165µM niclosamide oral dose, and only 0.65 micrograms for an intranasal 20µM niclosamide solution.

S1. Instrument Resolution (Photometric accuracy)
An expected solubility for niclosamide of only ~2μM in the low pH range corresponds to an absorbance of only 0.02 Absorbance units (AU). This is only an order of magnitude higher than the resolution (photometric accuracy) of the UV5nano which is quoted as +/-0.005 Absorbance units. Note: in the manual photometric accuracy is quoted < ± 0.01 A (potassium dichromate, Ph.Eur./USP method). It was also necessary to evaluate this resolution to make initial measurements of supernatant niclosamide concentration early on in the dissolution process of the crushed tablet powder. Resolution therefore was tested with nine samples in the same quartz cuvette (used in the same direction) which was washed and refilled for each series of three measurements As shown in Fig. S1A, (next page) are the Raw Spectra for the Blanks containing filtered Tris Buffered Saline Solution (TBSS) at pH 9.34. Three sets of blank spectra were taken (three consecutive measurements for each).

S2. Vanillin
In order to prove the material was vanillin an interesting side study was completed. 100µM vanillin solutions were made up in pH 8.35 and 9.35 Tris buffer and serially diluted to provide calibration spectra for comparison with the Yomesan spectra. As shown in Fig. S1A and B, the UV-Vis spectra of vanillin at pH 8.35 and 9.35 in Tris buffer have a single peak at ~347nm. The spectra and peak agreed well with the experiments of Shu et al [1], and their test spectra, shown in Fig  S1D, of vanillin in 10mM sodium borate at pH 8.0, and an absorption maximum at λmax of 347 nm. Interestingly, at pH 7.0, as shown in Fig. S2, the vanillin spectrum showed two absorbance peaks at 316nm and 347nm, consistent with that reported by others including NIST [2] (pH was not given but assumed to be water). This side study on vanillin was important because it identified the "impurity" in the Yomesan spectrum as vanillin and has actually provided confirming data, newly presented together with vanillin's own pH-dependence, and the spectra that can be subtracted from the Yomesan dissolution spectra to reveal the niclosamide in supernatant solution.
Plotting the absorbance of the 347nm peak versus concentration in Fig. S2C, shows that these two separate samples, made up in two different buffer solutions, one at pH 9.35 and the other at pH 8.35 give a linear (calibration) relationship and agree fairly well, with slopes of 40.8µM/AU and 40.5 µM/AU. For completion, because Yomesan was also tested at ~pH 7, the impurity spectrum would also need to be accounted for there too. As shown in Fig. S3A, vanillin at pH 7.06 actually has a completely different serial dilution spectrum to the higher pH spectra. The spectra at pH 7.06 has two absorbance peaks at 316nm and 347nm and are consistent with those reported by others including NIST [2] (pH is not given but assumed to be water).
Interestingly, then, like niclosamide, vanillin is also a weak acid. As reported by Shu et al, [1] the pKa value of vanillin is 7.38, which is actually quite close to that of niclosamide of 7.12 as derived earlier for pH dependent solubility data [3]. As also shown in Fig S3B, at a pH of 8.35, that is above vanillin's pKa of 7.38, the 317nm peak all but disappears as the more dominant, presumably, salt peak at 347 increases with increasing domination of the negatively charged salt. Thus, when the pH is >7.38, vanillin in solution is increasingly negatively charged and as dictated by weak acid equilibria and the Henderson Hasselbalch equation, will have a higher total solubility than when the pH is <7.38, where it is protonated, in a neutral state and less soluble. While the pHs were not specified, the solubility of vanillin has been reported to be 67.65mM [4], 72.3mM (Yalkowski, [5]) and calculated, at pH 7.06, by MarvinSketch (Chem axon) to be 129mM. Thus, at these concentrations of 100uM and less, both the weak acid and the deprotonated salt are expected to be water soluble. This side study on vanillin was important because it identified the "impurity" in the Yomesan spectrum as vanillin and has actually provided confirming data, newly presented together of vanillin's own pH dependence, and the spectra that can be subtracted from the Yomesan dissolution spectra to reveal the niclosamide in supernatant solution.

S3. Talc and Cornstarch
For completion, shown in Fig. S4 are the spectra of saturated solutions of talc and cornstarch in pH 7 tris buffer. given that common excipients used as binders and disintegrants in oral tablets include talc (USP Medisca) and cornstarch (supplier), in all tablets. Although not listed on the packaging, noting that the tablet weight of the 500mg niclosamide was almost doubled by the excipients, and that and talc was especially evident in optical microscope images of the Niclosig, spectra of saturated solutions of talc and cornstarch were obtained, as shown here. Both talc (290nm -410nm) and cornstarch (290nm -400nm) do appear to absorb in the same range as niclosamide. However, as can be appreciated, even the saturated solutions of these USP excipients, give a very low averaged absorbances, ~0.004 and 0.005 respectively in the same range as the resolution of the spectrometer (0.005A). Nevertheless, given the apparently high talc amount that would provide a saturated solution, the talc spectra were subtracted from the Niclosig absorbances when made in pH 7.0 Tris Buffer.
For completion, here are the spectra of saturated solutions of talc and cornstarch in pH 7 tris buffer.
given that common excipients used as binders and disintegrants in oral tablets include talc (USP Medisca) and cornstarch (supplier), in all tablets. Although not listed on the packaging, noting that the tablet weight of the 500mg niclosamide was almost doubled by the excipients, and that and talc was especially evident in optical microscope images of the Niclosig, spectra of saturated solutions of talc and cornstarch were obtained, as shown here. Both talc (290nm -410nm) and cornstarch (290nm -400nm) do appear to absorb in the same range as niclosamide. However, as can be appreciated, even the saturated solutions of these USP excipients, give a very low averaged absorbances, ~0.004 and 0.005 respectively in the same range as the resolution of the spectrometer (0.005A). Nevertheless, given the apparently high talc amount that would provide a saturated solution, the talc spectra were subtracted from the Niclosig absorbances when made in pH 7.0 Tris Buffer.

S4. Fraction of Niclosamide Extracted from Yomesan Powder
While it is expected that niclosamide of a given polymorph would have a consistent concentration at a given pH, (as reported earlier), the supernatant niclosamide concentration extracted from the tablet material may not necessarily reach, or fully represent, that concentration. For completion and to give the numbers as to what might be expected in a scaled-up extraction, Fig. S5A and B show the measured peak maximum niclosamide concentrations and % niclosamide that were extracted by dissolution of Yomesan tablet powder in nominal pH 9.35 Tris buffer. As can be seen in Fig. S5A none of the samples reached the expected value measured for the anhydrous AK Sci niclosamide in the previous publication, [3], which at pH 9.22 was 344µM. This could reflect some drug binding within the tablet excipient materials (cornstarch, talc, …), as well as the conversion to the lower solubility polymorph.
As discussed in the main text, in association with the morphological evidence by optical microscopy, it seems clear that niclosamide used in the tablets is a mixture of anhydrous and monohydrate, and it is the conversion or growth of monohydrate crystals on the anhydrous aggregates that results in the lowering of the supernatant concentration. Thus, taking these three concentration results together, as shown by the black filled symbols, in Fig. S5A, the extraction of niclosamide from Yomesan tablet powder increases, as might be expected with increasing amounts of niclosamide added. However, even for 1mM niclosamide equivalent (~3x excess material) it does not achieve the expected pH-dependent concentration of 344µM that a pure niclosamide sample does of an anhydrous niclosamide (AK Sci) at the same pH of 9.22, as measured earlier. In Fig. S5B, the same data is plotted now as a percent of niclosamide that is extracted from the given amount of added Yomesan powder, as an equivalent total niclosamide concentration. As the graph shows, increasing the amount of available niclosamide in the added suspended powder does not proportionately increase the amount of niclosamide that can be extracted; in fact, the extracted yield% actually decreases and so behaves with diminishing returns. What this data shows then is that niclosamide can certainly be extracted from Yomesan tablets into nominally pH 9.35 Tris buffer achieving concentrations in the 170µM to 260µM range. However, with a faster rate of dissolution for the greater amounts available (larger surface area of more material) and therefore increased concentration in supernatant solution, it appears that the excess material initiates the growth of the lower solubility polymorph that starts to bring down the overall amount of niclosamide in supernatant solution. Thus, to reiterate, some niclosamide either remains bound to one or more excipients, and/or the growth of the lower solubility monohydrate polymorphs depletes the supernatant niclosamide concentration.

S5. Initial Rates of Dissolution
It is also clear from the solid straight lines in Figs. 4 and 5 (main text) that the initial rates of dissolution increase as more powder is available, i.e., 1mM Yomesan niclosamide dissolves the fastest and the 300µM dissolves the slowest. This is quantified in the next two graphs in Fig. S6.
Measurements of supernatant niclosamide concentration for the initial dissolution of Yomesan niclosamide over the first few minutes give the initial dissolution rates, as shown in Fig. S6A. The slopes of the lines in Fig. S6A are plotted in Fig. S6B, as the amount of niclosamide dissolved out of the tablet material per minute (basically dm/dt). Initial rates over the first 5, 10 and 15 mins were: 14.4µM/min for 1mM; 7.6µM/min for 600µM; and 4.2µM/min for 300µM. Although not known exactly, surface area and mixing were constant for each sample, i.e., the dissolution was carried out using the same ground powder and hence consistent surface area and the same stirring speed of 300rpm for the same stir bar. The initial rate of dissolution increases with increasing amount of niclosamide present, basically reflecting the increased surface area of material available to be dissolved. While only three points, the line does intercept at 0.0 with a linear relationship of 0.014 µM/min per µM of Yomesan-niclosamide equivalent added. Thus, as shown in these and the overall dissolution plots, we can expect that the more niclosamide available the faster it will increase in supernatant concentration. However, as shown next, more powder does not necessarily equate to a greater fraction of niclosamide being extracted, in fact it is quite the opposite.

S6. Niclosamide in Tris Buffered Saline Precipitates or Grows "Wheatsheaf" Crystal Morphologies
As additional context to the needle-shaped morphologies that are formed by growing on the particulate aggregates, it is interesting to compare other systems such as niclosamide precipitation into Tris Buffered Saline Solutions (TBSS). When pure niclosamide is precipitated from an ethanolic solution by solvent exchange into TBSS at pH 8.45, bright red wheatsheaf needle morphologies are rapidly A typical example is shown in Fig. S7. Needles (Fig. S7A) emanate from what appears to be a nucleating core shown at higher magnification (Fig. S7B). These precipitation experiments for niclosamide, including niclosamide sodium, will form the basis for a subsequent paper [28]. These data are very preliminary and motivate further pure niclosamide studies with alkali metal salts, where these red crystals are readily formed in the presence of sodium salts and buffers, but only in the higher pH range. The hypothesis to be tested would be that in order to form the sodium salt, there needs to be a considerable amount of the negatively charged niclosamide species (for the Nicand Na + interaction), and so this occurs best at the higher pH where niclosamide is almost fully deprotonated. The implication from this kind of data of any formulation of niclosamide in aqueous buffer is that, in the presence of alkali metal ions, niclosamide preparations at these slightly elevated pHs will move towards this most stable niclosamide-sodium state. Even here though, at 10µM there is 1,000 times the IC100 for preventing viral infection of nasal and bronchial epithelial cells [6].

S7. Comparative UV-Vis Spectra
Because Yomesan contained vanillin, the UV-Vis spectrum of vanillin needed to be subtracted from all subsequent spectra for the Yomesan-niclosamide dissolution. Presented here for completion are the typical baseline-corrected spectra and the 30s-subtracted spectra for the two main Specific Aims: The Concentrations at pH 9.35 and the pH dependence. Only the consecutive spectra where there is increasing concentration are shown.

S7.1 Concentration Dependence
The concentration dependence for the dissolution of niclosamide from Yomesan powder was carried out at pH 9.35 at 300µM, 600µM and 1mM initial added Yomesan-niclosamide equivalent concentrations. Shown in Fig. S8 are the three series of UV-Vis spectra that generated the supernatant niclosamide concentrations plotted in the main text in Figs 4, 5 and 6. In the left-hand column, are the baseline-corrected spectra, and in the right-hand column are the same data but with the 30s spectrum subtracted from all subsequent time points. The 30s spectrum is shown as a dashed line for comparison and to show the effect of subtracting what is essentially the vanillin spectrum.
300µM Fig. S8A At 300µM added Yomesan niclosamide-equivalent, absorbance spectra for all 180mins progressively increase in intensity for each time point. There was actually no peak or decrease and as shown in Fig. 4A (main text), all points up to 180mins were used to give a logarithmic fit. The absorbances and hence niclosamide concentrations did not max-out the instrument. A shown in the right-hand spectra the 30s spectrum (orange dashed line) were subtracted to give the 333nm values reported as niclosamide concentrations versus time after addition of the Yomesan powder in Fig.4A. 600µM Fig. S8B At 600µM added Yomesan niclosamide-equivalent, baseline corrected absorbance spectra shown in the left progressively increased in intensity until ~ 60 mins when the absorbances started to reach the limit of the instrument. The spectra became noisy, but they average out to values that were similar to the 2x dilution of the same samples. Thus, the limits of the instrument were approached but not exceeded. The 30s spectrum (orange line) at this higher initial Yomesan concentration did increase (doubled compared to the 300µM sample) as also shown in Fig. 2A and B (main text). Subtracting this 30s spectrum (orange dashed line) gave the series of spectra on the right, that increased in intensity out to 150 mins. These spectra were thus baseline-corrected, 30ssubtracted and 2x-diluted from the 50min time point onwards and provide the niclosamide concentrations for Fig 4B (main text).
1mM Fig. S8C The baseline corrected spectra for the 1mM sample reached the instrument maximum after only 20 mins of dissolution. As shown in Fig. 4C (main text) the values plateaued, while the 2x diluted samples continued to show the increasing absorbance. They reached a peak after 90mins and then the absorbances started to decrease. Thus, only the first 90 mins are shown as increasing absorbance. Again, the amount of vanillin that came with this higher 1mM Yomesan niclosamide-equivalent concentration, is reflected in the absorbance intensity of the 30s spectrum (orange line) and data on Fig. 2A and B (main text). Again, subtracting this 30s spectrum gave the baseline corrected, 30s subtracted and 2x diluted spectra form which the data in Fig. 4C (main text) was derived. Fig S8. UV-Vis spectra for dissolution of niclosamide at initial added Yomesan-niclosamideequivalent concentrations of A) 300µM, B) 600µM and C) 1mM in pH 9.35 Tris Buffer. (Left) are the baseline corrected spectra; (right) 30s-subtracted spectra showing the 30s impurity spectrum in dashed orange. For 600µM and 1mM, the spectra that reach the limit of the Spectrometer are diluted 2x then absorbance is doubled. (NOTE: different y-axis scales are used to accommodate the data)